US20150056199A1 - Tgf-beta receptor type ii variants and uses thereof - Google Patents

Tgf-beta receptor type ii variants and uses thereof Download PDF

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US20150056199A1
US20150056199A1 US14/465,182 US201414465182A US2015056199A1 US 20150056199 A1 US20150056199 A1 US 20150056199A1 US 201414465182 A US201414465182 A US 201414465182A US 2015056199 A1 US2015056199 A1 US 2015056199A1
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seq
tβrii
amino acid
polypeptide
acid sequence
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Ravindra Kumar
Asya Grinberg
Dianne S. Sako
Roselyne Castonguay
Rita Steeves
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Acceleron Pharma Inc
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Acceleron Pharma Inc
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Priority to US14/465,182 priority Critical patent/US20150056199A1/en
Assigned to ACCELERON PHARMA, INC. reassignment ACCELERON PHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KUMAR, RAVINDRA, GRINBERG, ASYA, CASTONGUAY, Roselyne, SAKO, DIANNE S., STEEVES, RITA
Publication of US20150056199A1 publication Critical patent/US20150056199A1/en
Priority to US15/044,883 priority patent/US9809637B2/en
Priority to US15/714,015 priority patent/US10316076B2/en
Priority to US16/393,277 priority patent/US10981973B2/en
Priority to US16/777,546 priority patent/US11008377B2/en
Priority to US17/229,132 priority patent/US20210230252A1/en
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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    • C07K2317/00—Immunoglobulins specific features
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • TGF ⁇ transforming growth factor-beta
  • BMP bone morphogenetic proteins
  • OP osteogenic proteins
  • GDF growth and differentiation factors
  • MIS mullerian inhibitory substances
  • GDNF glial derived neurotrophic factors
  • TGF ⁇ superfamily members transduce their signals across the plasma membrane by inducing the formation of heteromeric complexes of specific type I and type II serine/threonine kinase receptors, which in turn activate a particular subset of SMAD proteins (some inhibitory and some excitatory).
  • SMAD proteins include SMAD proteins, and others excitatory proteins.
  • the SMAD molecule compounds relay the signals into the nucleus where they direct transcriptional responses in concert with other proteins.
  • Dysfunctional TGF ⁇ superfamily signaling has been linked to several clinical disorders including cancer, fibrosis, bone diseases, diabetic nephropathy, as well as chronic vascular diseases such as atherosclerosis.
  • compositions and methods for modulating TGF ⁇ superfamily signaling are provided.
  • the disclosure provides T ⁇ RII polypeptides and the use of such T ⁇ RII polypeptides as selective antagonists for GDF15, TGF ⁇ 1 or TGF ⁇ 3.
  • polypeptides comprising part or all of the T ⁇ RII extracellular domain (ECD), with or without additional mutations, bind to and/or inhibit GDF15, TGF ⁇ 1 or TGF ⁇ 3 with varying affinities.
  • ECD extracellular domain
  • the disclosure provides T ⁇ RII polypeptides for use in selectively inhibiting TGF ⁇ superfamily associated disorders.
  • the disclosure provides polypeptides comprising mutations and/or truncations in the extracellular domain of T ⁇ RII.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence from the extracellular domain of T ⁇ RII and a heterologous amino acid sequence, wherein the first amino acid sequence comprises or consists of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to a) a sequence beginning at any of positions 23 to 35 of SEQ ID NO: 5 and ending at any of positions 153 to 159 of SEQ ID NO: 5 or b) a sequence beginning at any of positions 23 to 60 of SEQ ID NO: 6 and ending at any of positions 178 to 184 of SEQ ID NO: 6.
  • the disclosure provides polypeptides comprising a wild-type or altered and/or truncated extracellular domain of T ⁇ RII fused to at least a portion of the Fc domain of a human IgG2.
  • a T ⁇ RII fusion polypeptide comprising a first amino acid sequence from the extracellular domain of T ⁇ RII and a heterologous amino acid sequence, wherein the first amino acid sequence comprises or consists of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to a) a sequence beginning at any of positions 23 to 35 of SEQ ID NO: 5 and ending at any of positions 153 to 159 of SEQ ID NO: 5 or b) a sequence beginning at any of positions 23 to 60 of SEQ ID NO: 6 and ending at any of positions 178 to 184 of SEQ ID NO: 6, and wherein the polypeptide comprises a second polypeptide sequence
  • SEQ ID NO:50 is encoded by the nucleic acid sequence of SEQ ID NO:51.
  • the disclosure provides polypeptides with an amino acid sequence that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%. 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:50.
  • the disclosure provides polypeptides that are encoded by a nucleic acid sequence that comprises or consists of a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%. 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:51.
  • the first amino acid sequence comprises or consists of the sequence beginning at position 23 of SEQ ID NO: 5 and ending at position 159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 29 of SEQ ID NO: 5 and ending at position 159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 35 of SEQ ID NO: 5 and ending at position 159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 23 of SEQ ID NO: 5 and ending at position 153 of SEQ ID NO: 5.
  • the first amino acid sequence comprises or consists of the sequence beginning at position 29 of SEQ ID NO: 5 and ending at position 153 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 35 of SEQ ID NO: 5 and ending at position 153 of SEQ ID NO: 5.
  • the first amino acid sequence comprises or consists of the sequence beginning at position 23 of SEQ ID NO: 6 and ending at positions 184 of SEQ ID NO: 6. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 29 of SEQ ID NO: 6 and ending at position 184 of SEQ ID NO: 6. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 23 of SEQ ID NO: 6 and ending at position 178 of SEQ ID NO: 6. In some embodiments, the first amino acid sequence comprises or consists of the sequence beginning at position 29 of SEQ ID NO: 6 and ending at position 178 of SEQ ID NO: 6.
  • the first amino acid sequence comprises or consists of a sequence that has a D at the position corresponding to position 36 of SEQ ID NO: 47 and/or a K at the position corresponding to position 76 of SEQ ID NO: 47.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or identical to the sequence of SEQ ID NO: 7 or SEQ ID NO: 13, or active fragment thereof, and a second heterologous portion, wherein the first amino acid sequence has a D at the position corresponding to position 36 of SEQ ID NO: 47 and/or a K at the position corresponding to position 76 of SEQ ID NO: 47.
  • the first amino acid sequence comprises an N-terminal truncation of 1-12 amino acids corresponding to amino acids 1-12 of SEQ ID NO: 7 or 1-37 amino acids corresponding to amino acids 1-37 of SEQ ID NO: 13. In some embodiments, the first amino acid sequence comprises an N-terminal truncation of 6 amino acids corresponding to amino acids 1-6 of SEQ ID NO: 7 or SEQ ID NO: 13. In some embodiments, the first amino acid sequence comprises an N-terminal truncation of 12 amino acids corresponding to amino acids 1-12 of SEQ ID NO: 7 or 37 amino acids corresponding to amino acids 1-37 of SEQ ID NO: 13.
  • the first amino acid sequence comprises a C-terminal truncation of 1-6 amino acids corresponding to amino acids 137-132 of SEQ ID NO: 7 or amino acids 162-157 of SEQ ID NO: 13. In some embodiments, the first amino acid sequence comprises a C-terminal truncation of 6 amino acids corresponding to amino acids 132-137 of SEQ ID NO: 7 or amino acids 157-162 of SEQ ID NO: 13. In some embodiments, the first amino acid sequence comprises an insertion corresponding to SEQ ID NO: 18 between the residues corresponding to positions 117 and 118 of SEQ ID NO: 47.
  • the heterologous portion comprises one or more polypeptide portions that enhance one or more of: in vivo stability, in vivo half life, uptake/administration, tissue localization or distribution, formation of protein complexes, and/or purification.
  • the heterologous portion comprises a polypeptide portion selected from: an immunoglobulin Fc domain and a serum albumin.
  • the immunoglobulin Fc domain is joined to the T ⁇ RII polypeptide by a linker.
  • the polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
  • the polypeptide is glycosylated.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of T ⁇ RII that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and a second heterologous portion.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of T ⁇ RII that comprises an amino acid sequence that is at least 96% identical to an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and a second heterologous portion.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of T ⁇ RII that comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and a second heterologous portion.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of T ⁇ RII that comprises an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and a second heterologous portion.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of T ⁇ RII that comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49, and a second heterologous portion.
  • the disclosure provides a T ⁇ RII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of T ⁇ RII that comprises an amino acid sequence is an amino acid sequence selected from SEQ ID NOs: 7-17 and 47-49 and a second heterologous portion.
  • the disclosure provides a polypeptide comprising or consisting of an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereof with the leader sequence removed, e.g., a polypeptide comprising or consisting of an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.
  • the disclosure provides a polypeptide comprising or consisting of an amino acid sequence that is at least 96% identical to an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereof with the leader sequence removed, e.g., a polypeptide comprising or consisting of an amino acid sequence that is at least 96% identical to an amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.
  • the disclosure provides a polypeptide comprising or consisting of an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereof with the leader sequence removed, e.g., a polypeptide comprising or consisting of an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.
  • the disclosure provides a polypeptide comprising or consisting of an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereof with the leader sequence removed, e.g., a polypeptide comprising or consisting of an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.
  • the disclosure provides a polypeptide comprising or consisting of an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereof with the leader sequence removed, e.g., a polypeptide comprising or consisting of an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.
  • the disclosure provides a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or the portion thereof with the leader sequence removed, e.g., a polypeptide comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.
  • the disclosure provides a T ⁇ RII polypeptide comprising of an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a complement of a nucleotide sequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the T ⁇ RII polypeptide may be selected that it does not include a full-length T ⁇ RII ECD.
  • a T ⁇ RII polypeptide may be used as a monomeric protein or in a dimerized form.
  • a T ⁇ RII polypeptide may also be fused to a second polypeptide portion to provide improved properties, such as increased half-life or greater ease of production or purification.
  • a fusion may be direct or a linker may be inserted between the T ⁇ RII polypeptide and any other portion.
  • a linker may be structured or unstructured and may consist of 1, 2, 3, 4, 5, 10, 15, 20, 30, 50 or more amino acids, optionally relatively free of secondary structure.
  • a T ⁇ RII polypeptide of the disclosure binds human GDF15 with an equilibrium dissociation constant (K D ) less than 1 ⁇ 10 ⁇ 8 M.
  • a T ⁇ RII polypeptide of the disclosure has a glycosylation pattern characteristic of expression of the polypeptide in CHO cells.
  • the disclosure provides a homodimer comprising two T ⁇ RII polypeptides of the disclosure.
  • the disclosure provides an isolated polynucleotide comprising a coding sequence for the T ⁇ RII polypeptides of the disclosure. In some embodiments, the disclosure provides a recombinant polynucleotide comprising a promoter sequence operably linked to the isolated polynucleotide. In some embodiments, the disclosure provides a cell transformed with an isolated polynucleotide or a recombinant polynucleotide of the disclosure. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell or a human cell. In some embodiments, the cell is an HEK-293 cell.
  • the disclosure provides a pharmaceutical preparation comprising the T ⁇ RII polypeptides or homodimers of the disclosure and a pharmaceutically acceptable excipient.
  • the disclosure provides a method of modulating the response of a cell to a TGF ⁇ superfamily member, the method comprising exposing the cell to a T ⁇ RII polypeptide or homodimer of the disclosure.
  • the disclosure provides a method of treating a disease or condition associated with a TGF ⁇ superfamily member in a patient in need thereof, the method comprising administering to the patient an effective amount of the T ⁇ RII polypeptides or homodimers of the disclosure.
  • the TGF ⁇ superfamily member is TGF ⁇ 1, TGF ⁇ 3 or GDF15.
  • the disease or condition is a cancer.
  • the cancer is selected from stomach cancer, intestinal cancer, skin cancer, breast cancer, melanoma, bone cancer and thyroid cancer.
  • the disease or condition is a fibrotic or sclerotic disease or disorder.
  • the fibrotic or sclerotic disease or disorder is selected from scleroderma, atherosclerosis, liver fibrosis, diffuse systemic sclerosis, glomerulonephritis, neural scarring, dermal scarring, radiation-induced fibrosis, hepatic fibrosis, and myelofibrosis.
  • the disease or condition is heart disease.
  • the disease or condition is selected from hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, pre-eclampsia, atherosclerosis, restenosis, and hypertrophic cardiomyopathy/congestive heart failure.
  • HHT hereditary hemorrhagic telangiectasia
  • Marfan syndrome Loeys-Dietz syndrome
  • familial thoracic aortic aneurysm syndrome familial thoracic aortic aneurysm syndrome
  • arterial tortuosity syndrome pre-eclampsia
  • atherosclerosis atherosclerosis
  • restenosis restenosis
  • hypertrophic cardiomyopathy/congestive heart failure hypertrophic cardiomyopathy/congestive heart failure.
  • the disclosure provides an antibody, or antigen binding fragment thereof, that binds to GDF15 and blocks the interaction between GDF15 and T ⁇ RII.
  • the disclosure provides a GDF15 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof that binds T ⁇ RII, wherein the GDF15 polypeptide is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure, with respect to protein contaminants.
  • the disclosure provides a GDF15 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof that binds to a T ⁇ RII polypeptide of the disclosure, wherein the GDF15 polypeptide is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure, with respect to protein contaminants.
  • the GDF15 polypeptide binds T ⁇ RII with an equilibrium dissociation constant (K D ) of no greater than 10 ⁇ 8 M. In some embodiments, the GDF15 polypeptide binds to a T ⁇ RII polypeptide of the disclosure with an equilibrium dissociation constant (K D ) of no greater than 10 ⁇ 8 M.
  • the GDF15 polypeptide is produced by expression in CHO cells.
  • the disclosure provides a method of concentrating or purifying GDF15, comprising contacting a sample containing GDF15 with a T ⁇ RII polypeptide of the disclosure.
  • FIG. 1 shows the amino acid sequence of native precursor for human GDF15 (NCBI reference seq: NP — 004855.2).
  • Solid underline indicates mature GDF15 (residues 197-308), with N-terminus determined by sequencing. Dotted underline denotes leader (residues 1-29).
  • FIG. 2 shows a nucleotide sequence encoding native precursor for human GDF15.
  • Solid underline indicates the sequence encoding mature GDF15 (nucleotides 589-924), and dotted underline denotes the sequence encoding the leader (nucleotides 1-87).
  • a silent mutation (G456A) used to disrupt a SfoI site in NM — 004864.2 is double underlined.
  • FIG. 3 shows the amino acid sequence of native precursor for murine GDF15 (NP — 035949.2). Solid underline indicates mature GDF15 (residues 192-303), with N-terminus determined by sequencing. Dotted underline denotes leader (residues 1-30).
  • FIG. 4 shows a nucleotide sequence encoding native precursor for murine GDF15 (derived from NM — 011819.2). Solid underline indicates the sequence encoding mature GDF15 (nucleotides 574-909), and dotted underline denotes the sequence encoding the leader (nucleotides 1-90).
  • FIG. 5 shows the amino acid sequence of native precursor for the B (short) isoform of human TGF ⁇ receptor type II (hT ⁇ RII) (NP — 003233.4).
  • Solid underline indicates the mature extracellular domain (ECD) (residues 23-159), and double underline indicates valine that is replaced in the A (long) isoform. Dotted underline denotes leader (residues 1-22).
  • FIG. 6 shows the amino acid sequence of native precursor for the A (long) isoform of human T ⁇ RII (NP — 001020018.1).
  • Solid underline indicates the mature ECD (residues 23-184), and double underline indicates the splice-generated isoleucine substitution. Dotted underline denotes leader (residues 1-22).
  • FIG. 7 shows N-terminal alignment of hT ⁇ RII short truncations and their hT ⁇ RII long counterparts.
  • the 25-amino-acid insertion present in hT ⁇ RII long truncations is underlined. Note that the splicing process causes the valine flanking the insertion site in the short isoform to be replaced by an isoleucine in the long isoform. Boxed sequence denotes leader.
  • Proteins described herein are the human forms, unless otherwise specified. NCBI references for the proteins are as follows: human T ⁇ RII isoform A (hT ⁇ RII long ), NP — 001020018.1; human T ⁇ RII isoform B (hT ⁇ RII short ), NP — 003233.4; human GDF15, NP — 004855.2; murine GDF15, NP — 035949.2. Sequences of native T ⁇ RII and GDF15 proteins from human and mouse are set forth in FIGS. 1-6 .
  • the TGF ⁇ superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. By manipulating the activity of a member of the TGF ⁇ family, it is often possible to cause significant physiological changes in an organism.
  • GDF8 also called myostatin
  • inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.
  • TGF ⁇ signals are mediated by heteromeric complexes of type I (e.g. T ⁇ RI) and type II (e.g. T ⁇ RII) serine/threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins upon ligand stimulation (Massagué, 2000, Nat. Rev. Mol. Cell. Biol. 1:169-178).
  • type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity.
  • Type I receptors are essential for signaling; and type II receptors are required for binding ligands and for expression of type I receptors.
  • Type I and II receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
  • TGF ⁇ has three mammalian isoforms, TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3, each with distinct functions in vivo.
  • the binding of TGF ⁇ s to T ⁇ RII is a crucial step in initiating activation of the TGF ⁇ signaling pathway, leading to phosphorylation of SMAD2, and translocation of the activated SMAD2/SMAD4 complex to the nucleus to modulate gene expression.
  • GDF15 Growth differentiation factor 15
  • TGF ⁇ TGF ⁇ family
  • prodomain a prodomain that is removed through cleavage by a furin-like protease at the canonical RXXR site to generate mature dimeric GDF15.
  • GDF15 has been described in the literature as macrophage inhibitory cytokine-1 (MIC-1), placental bone morphogenic protein (PLAB), placental transforming growth factor beta (PTGF ⁇ ), prostate derived factor (PDF), and non-steroidal anti-inflammatory activated gene-1 (NAG-1) reflecting the different functions that have been implied for this protein.
  • GDF15 has been linked to several physiologic and pathologic conditions. For example, GDF15 is highly expressed in the placenta, and is necessary for the maintenance of pregnancy. GDF15 concentration is also notably increased in the serum of patients with prostate, colorectal, or pancreatic cancer, as well as glioma. GDF15 has not been shown biochemically to bind or interact directly with any receptor.
  • the present disclosure relates in part to the discovery that the TGF ⁇ type II receptor, T ⁇ RII, binds to GDF15 with high affinity and is a functional receptor for GDF15.
  • T ⁇ RII fusion polypeptides, and other polypeptides containing a ligand-binding portion of T ⁇ RII are demonstrated herein to inhibit GDF15-induced gene activation.
  • the potent inhibition of GDF15 signaling provides evidence that T ⁇ RII is a functional type II receptor for GDF15, opening a new avenue for therapeutic interventions in this signaling pathway. Therefore, in part, the disclosure identifies a physiological, high-affinity receptor for GDF15 polypeptides.
  • T ⁇ RII is the known type II receptor for TGF ⁇ and binds with high affinity to TGF ⁇ 1 and TGF ⁇ 3.
  • Human T ⁇ RII occurs naturally in at least two isoforms—A (long) and B (short)—generated by alternative splicing in the extracellular domain (ECD) ( FIGS. 6 and 5 and SEQ ID NOS: 6 and 5).
  • the long isoform has a 25-amino-acid insertion and the splicing process causes the valine flanking the insertion site in the short isoform to be replaced by an isoleucine in the long isoform.
  • Soluble receptor ectodomains can function as scavengers or ligand traps to inhibit ligand-receptor interactions.
  • Ligand traps such as soluble T ⁇ RII-Fc fusion proteins incorporating the native T ⁇ RII extracellular domain (ectodomain) will function as pan-inhibitors against T ⁇ RII ligands, including, TGF ⁇ 1, TGF ⁇ 3 and based on the findings disclosed herein, GDF15. While in some therapeutic settings this broader spectrum of ligand-binding and signal inhibition may be advantageous, in other settings a more selective molecule may be superior. It is highly desirable for ligand traps such as T ⁇ RII ectodomain polypeptides to exhibit selective ligand-binding profiles.
  • the present disclosure relates to the surprising discovery that polypeptides comprising a truncated portion of the extracellular domain of T ⁇ RII and/or mutations within the extracellular domain have differential inhibitory effects on cell signaling by GDF15, TGF ⁇ 1 or TGF ⁇ 3.
  • the disclosure provides ligand traps, generated by a series of mutations and/or truncations in the extracellular domain of T ⁇ RII, that exhibit varying ligand-binding profiles distinct from that of the native T ⁇ RII extracellular domain.
  • the variant T ⁇ RII polypeptides disclosed herein provide advantageous properties relative to the native full-length extracellular domain, and may be used to selectively inhibit pathways mediated by the different T ⁇ RII ligands in vivo.
  • the disclosure provides T ⁇ RII polypeptides as antagonists of GDF15, TGF ⁇ 1 or TGF ⁇ 3 for use in treating various GDF15-, TGF ⁇ 1- or TGF ⁇ 3-associated disorders. While not wishing to be bound to any particular mechanism of action, it is expected that such polypeptides act by binding to GDF15, TGF ⁇ 1 or TGF ⁇ 3 and inhibiting the ability of these ligands to form ternary signaling complexes.
  • Naturally occurring T ⁇ RII proteins are transmembrane proteins, with a portion of the protein positioned outside the cell (the extracellular portion) and a portion of the protein positioned inside the cell (the intracellular portion). Aspects of the present disclosure encompass variant T ⁇ RII polypeptides comprising mutations within the extracellular domain and/or truncated portions of the extracellular domain of T ⁇ RII. As described above, human T ⁇ RII occurs naturally in at least two isoforms—A (long) and B (short)—generated by alternative splicing in the extracellular domain (ECD) ( FIGS. 6 and 5 and SEQ ID NOS: 6 and 5).
  • SEQ ID NO: 7 which corresponds to residues 23-159 of SEQ ID NO: 5, depicts the native full-length extracellular domain of the short isoform of T ⁇ RII.
  • SEQ ID NO: 13 which corresponds to residues 23-184 of SEQ ID NO: 6, depicts the native full-length extracellular domain of the long isoform of T ⁇ RII.
  • amino acid position numbering with regard to variants based on the T ⁇ RII short and long isoforms refers to the corresponding position in the native precursors, SEQ ID NO: 5 and SEQ ID NO:6, respectively.
  • T ⁇ RII polypeptides may bind to and inhibit the function of a TGF ⁇ superfamily member, such as but not limited to, GDF15, TGF ⁇ 1 or TGF ⁇ 3.
  • T ⁇ RII polypeptides may include a polypeptide consisting of, or comprising, an amino acid sequence at least 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a truncated ECD domain of a naturally occurring T ⁇ RII polypeptide, whose C-terminus occurs at any of amino acids 153-159 of SEQ ID NO: 5.
  • T ⁇ RII polypeptides may include a polypeptide consisting of, or comprising, an amino acid sequence at least 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a truncated ECD domain of a naturally occurring T ⁇ RII polypeptide, whose C-terminus occurs at any of amino acids 178-184 of SEQ ID NO: 6.
  • a T ⁇ RII polypeptide does not include more than 5 consecutive amino acids, or more than 10, 20, 30, 40, 50, 52, 60, 70, 80, 90, 100, 150 or 200 or more consecutive amino acids from a sequence consisting of amino acids 160-567 of SEQ ID NO: 5 or from a sequence consisting of amino acids 185-592 of SEQ ID NO: 6.
  • the unprocessed T ⁇ RII polypeptide may either include or exclude any signal sequence, as well as any sequence N-terminal to the signal sequence.
  • the N-terminus of the mature (processed) T ⁇ RII polypeptide may occur at any of amino acids 23-35 of SEQ ID NO: 5 or 23-60 of SEQ ID NO: 6.
  • mature T ⁇ RII polypeptides include, but are not limited to, amino acids 23-159 of SEQ ID NO: 5 (set forth in SEQ ID NO: 7), amino acids 29-159 of SEQ ID NO: 5 (set forth in SEQ ID NO: 9), amino acids 35-159 of SEQ ID NO: 5 (set forth in SEQ ID NO: 10), amino acids 23-153 of SEQ ID NO: 5 (set forth in SEQ ID NO: 11), amino acids 29-153 of SEQ ID NO: 5 (set forth in SEQ ID NO: 48), amino acids 35-153 of SEQ ID NO: 5 (set forth in SEQ ID NO: 47), amino acids 23-184 of SEQ ID NO: 6 (set forth in SEQ ID NO: 13), amino acids 29-184 of SEQ ID NO: 6 (set forth in SEQ ID NO: 15), amino acids 60-184 of SEQ ID NO:6 (set forth in SEQ ID NO: 10), amino acids 23-178 of SEQ ID NO: 6 (set forth in SEQ ID NO: 16), amino acids 29-178 of
  • a T ⁇ RII polypeptide may comprise a polypeptide that is encoded by nucleotides 73-465 of SEQ ID NO: 30, nucleotides 73-447 of SEQ ID NO: 34, nucleotides 73-465 of SEQ ID NO: 38, nucleotides 91-465 of SEQ ID NO: 38, or nucleotides 109-465 of SEQ ID NO: 38, or silent variants thereof or nucleic acids that hybridize to the complement thereof under stringent hybridization conditions (generally, such conditions are known in the art but may, for example, involve hybridization in 50% v/v formamide, 5 ⁇ SSC, 2% w/v blocking agent, 0.1% N-lauroylsarcosine, and 0.3% SDS at 65° C.
  • T ⁇ RII T ⁇ RII
  • corresponding variants based on the long isoform of T ⁇ RII will include nucleotide sequences encoding the 25-amino acid insertion along with a conservative Val-Ile substitution at the flanking position C-terminal to the insertion.
  • the T ⁇ RII polypeptides accordingly may include isolated extracellular portions of T ⁇ RII polypeptides, including both the short and the long isoforms, variants thereof (including variants that comprise, for example, no more than 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acid substitutions in the sequence corresponding to amino acids 23-159 of SEQ ID NO: 5 or amino acids 23-184 of SEQ ID NO: 6), fragments thereof, and fusion proteins comprising any of the foregoing, but in each case preferably any of the foregoing T ⁇ RII polypeptides will retain substantial affinity for at least one of GDF15, TGF ⁇ 1 or TGF ⁇ 3.
  • a T ⁇ RII polypeptide will be designed to be soluble in aqueous solutions at biologically relevant temperatures, pH levels, and osmolarity.
  • the variant T ⁇ RII polypeptides of the disclosure comprise one or more mutations in the extracellular domain that confer an altered ligand binding profile.
  • a T ⁇ RII polypeptide may include one, two, five or more alterations in the amino acid sequence relative to the corresponding portion of a naturally occurring T ⁇ RII polypeptide.
  • the mutation results in a substitution, insertion, or deletion at the position corresponding to position 70 of SEQ ID NO: 5.
  • the mutation results in a substitution, insertion, or deletion at the position corresponding to position 110 of SEQ ID NO: 5. Examples include, but are not limited to, an N to D substitution or a D to K substitution in the positions corresponding to positions 70 and 110, respectively, of SEQ ID NO: 5.
  • T ⁇ RII polypeptides include, but are not limited to, the sequences set forth in SEQ ID NO: 8, SEQ ID NO:14, SEQ ID NO: 12 and SEQ ID NO: 17.
  • a T ⁇ RII polypeptide may comprise a polypeptide or portion thereof that is encoded by nucleotides 73-483 of SEQ ID NO: 26, nucleotides 73-465 of SEQ ID NO: 42 or silent variants thereof or nucleic acids that hybridize to the complement thereof under stringent hybridization conditions.
  • the variant T ⁇ RII polypeptides of the disclosure further comprise an insertion of 36 amino acids (SEQ ID NO: 18) between the pair of glutamate residues (positions 151 and 152 of SEQ ID NO: 5, or positions 176 and 177 of SEQ ID NO: 6) located near the C-terminus of the human T ⁇ RII ECD, as occurs naturally in the human T ⁇ RII isoform C (Konrad et al., BMC Genomics 8:318, 2007).
  • T ⁇ RII polypeptides can be modified to selectively antagonize T ⁇ RII ligands.
  • Data presented here show that Fc fusion proteins comprising shorter N-terminally and C-terminally truncated variants of T ⁇ RII polypeptides display differential inhibitory effects on cellular signaling mediated by GDF15, TGF ⁇ 1 and TGF ⁇ 3.
  • N-terminally truncated variants beginning at amino acids 29 or 35 of SEQ ID NO: 5 and carrying, respectively, a 6- or 12-amino acid N-terminal truncation of the extracellular domain were found to inhibit GDF15 most potently, TGF ⁇ 3 least potently and TGF ⁇ 1 to an intermediate degree, compared to the full length extracellular domain of the short isoform of T ⁇ RII.
  • C-terminally truncated variants, ending at amino acid 153 of SEQ ID NO: 5 and carrying a 6-amino acid C-terminal truncation of the extracellular domain had no substantial effect on ligand binding and may therefore be used interchangeably with full length versions.
  • N to D substitution at the position corresponding to position 70 of SEQ ID NO: 5 was found to inhibit TGF ⁇ 3 potently, have intermediate effect on GDF15 and negligible effect on TGF ⁇ 1.
  • the N70 residue represents a potential glycosylation site.
  • an Fc fusion protein comprising a D to K substitution at the position corresponding to position 110 of SEQ ID NO: 5 was found to inhibit GDF15 most potently, TGF ⁇ 1 least potently and TGF ⁇ 3 to an intermediate degree compared to compared to the full length extracellular domain of the short isoform of T ⁇ RII.
  • the region around position 110 has not been associated with selectivity for the known T ⁇ RII ligands TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3.
  • T ⁇ RII polypeptides that contain mutations in the ECD, such as but not limited to, N70D and D110K (the numbering of the residues corresponds to that of SEQ ID NO: 5) and/or begin between amino acids 29 and 35 and/or terminate between amino acid 153 and amino acid 159 are all expected to be active and exhibit widely different inhibitory potencies towards the different ligands. Any of these truncated variant forms may be desirable to use, depending on the clinical or experimental setting.
  • a T ⁇ RII polypeptide binds to GDF15, and the T ⁇ RII polypeptide does not show substantial binding to TGF ⁇ 1 or TGF ⁇ 3. In certain embodiments, a T ⁇ RII polypeptide binds to TGF ⁇ 1, and the T ⁇ RII polypeptide does not show substantial binding to GDF15 or TGF ⁇ 3. In certain embodiments, a T ⁇ RII polypeptide binds to TGF ⁇ 3, and the T ⁇ RII polypeptide does not show substantial binding to GDF15 or TGF ⁇ 1. Binding may be assessed using purified proteins in solution or in a surface plasmon resonance system, such as a BiacoreTM system.
  • a T ⁇ RII polypeptide inhibits GDF15 cellular signaling, and the T ⁇ RII polypeptide has an intermediate or limited inhibitory effect on TGF ⁇ 1 or TGF ⁇ 3. In certain embodiments, a T ⁇ RII polypeptide inhibits TGF ⁇ 1 cellular signaling, and the T ⁇ RII polypeptide has an intermediate or limited inhibitory effect on GDF15 or TGF ⁇ 3. In certain embodiments, a T ⁇ RII polypeptide inhibits TGF ⁇ 3 cellular signaling, and the T ⁇ RII polypeptide has an intermediate or limited inhibitory effect on GDF15 or TGF ⁇ 1. Inhibitory effect on cell signaling can be assayed by methods known in the art.
  • an active portion of a T ⁇ RII polypeptide may comprise amino acid sequences 23-153, 23-154, 23-155, 23-156, 23-157, or 23-158 of SEQ ID NO: 5, as well as variants of these sequences starting at any of amino acids 24-35 of SEQ ID NO: 5.
  • an active portion of a T ⁇ RII polypeptide may comprise amino acid sequences 23-178, 23-179, 23-180, 23-181, 23-182, or 23-183 of SEQ ID NO: 6, as well as variants of these sequences starting at any of amino acids 24-60 of SEQ ID NO: 6.
  • T ⁇ RII polypeptides comprise amino acid sequences 29-159, 35-159, 23-153, 29-153 and 35-153 of SEQ ID NO: 5 or amino acid sequences 29-184, 60-184, 23-178, 29-178 and 60-178 of SEQ ID NO: 6. Variants within these ranges are also contemplated, particularly those having at least 80%, 85%, 90%, 95%, or 99% identity to the corresponding portion of SEQ ID NO: 5 or SEQ ID NO: 6.
  • a T ⁇ RII polypeptide may be selected that does not include the sequence consisting of amino acids 160-567 of SEQ ID NO:5 or amino acids 185-592 of SEQ ID NO:6.
  • the disclosure provides T ⁇ RII polypeptides sharing a specified degree of sequence identity or similarity to a naturally occurring T ⁇ RII polypeptide.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid “identity” is equivalent to amino acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com).
  • the following parameters are used in the GAP program: either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res.
  • Exemplary parameters include using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified, percent identity between two amino acid sequences is to be determined using the GAP program using a Blosum 62 matrix, a GAP weight of 10 and a length weight of 3, and if such algorithm cannot compute the desired percent identity, a suitable alternative disclosed herein should be selected.
  • the percent identity between two amino acid sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • Another embodiment for determining the best overall alignment between two amino acid sequences can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. ( Comp. App. Biosci., 6:237-245 (1990)).
  • a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity.
  • amino acid sequence identity is performed using the FASTDB computer program based on the algorithm of Brutlag et al. ( Comp. App. Biosci., 6:237-245 (1990)).
  • T ⁇ RII polypeptides may additionally include any of various leader sequences at the N-terminus. Such a sequence would allow the peptides to be expressed and targeted to the secretion pathway in a eukaryotic system. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783 (1992).
  • a native T ⁇ RII signal sequence may be used to effect extrusion from the cell.
  • Possible leader sequences include native leaders, tissue plasminogen activator (TPA) and honeybee mellitin (SEQ ID NOs. 22-24, respectively).
  • T ⁇ RII-Fc fusion proteins incorporating a TPA leader sequence examples include SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43. Processing of signal peptides may vary depending on the leader sequence chosen, the cell type used and culture conditions, among other variables, and therefore actual N-terminal start sites for mature T ⁇ RII polypeptides may shift by 1, 2, 3, 4 or 5 amino acids in either the N-terminal or C-terminal direction.
  • T ⁇ RII-Fc fusion proteins include SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62, as shown herein with the T ⁇ RII polypeptide portion underlined (see Examples). It will be understood by one of skill in the art that corresponding variants based on the long isoform of T ⁇ RII will include the 25-amino acid insertion along with a conservative Val-Ile substitution at the flanking position C-terminal to the insertion.
  • the present disclosure contemplates specific mutations of the T ⁇ RII polypeptides so as to alter the glycosylation of the polypeptide.
  • Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites.
  • Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (where “X” is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type T ⁇ RII polypeptide (for O-linked glycosylation sites).
  • a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
  • Another means of increasing the number of carbohydrate moieties on a T ⁇ RII polypeptide is by chemical or enzymatic coupling of glycosides to the T ⁇ RII polypeptide.
  • the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine.
  • Removal of one or more carbohydrate moieties present on a T ⁇ RII polypeptide may be accomplished chemically and/or enzymatically.
  • Chemical deglycosylation may involve, for example, exposure of the T ⁇ RII polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact.
  • Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118:131.
  • Enzymatic cleavage of carbohydrate moieties on T ⁇ RII polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350.
  • the sequence of a T ⁇ RII polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
  • T ⁇ RII polypeptides for use in humans will be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells are expected to be useful as well.
  • This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of a T ⁇ RII polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences.
  • the purpose of screening such combinatorial libraries may be to generate, for example, T ⁇ RII polypeptide variants which can act as either agonists or antagonist, or alternatively, which possess novel activities all together.
  • a variety of screening assays are provided below, and such assays may be used to evaluate variants.
  • a T ⁇ RII polypeptide variant may be screened for ability to bind to a T ⁇ RII ligand, to prevent binding of a T ⁇ RII ligand to a T ⁇ RII polypeptide or to interfere with signaling caused by a T ⁇ RII ligand.
  • the activity of a T ⁇ RII polypeptide or its variants may also be tested in a cell-based or in vivo assay, particularly any of the assays disclosed in the Examples.
  • Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a T ⁇ RII polypeptide comprising an extracellular domain of a naturally occurring T ⁇ RII polypeptide.
  • mutagenesis can give rise to variants which have serum half-lives dramatically different than the corresponding wild-type T ⁇ RII polypeptide.
  • the altered protein can be rendered either more stable or less stable to proteolytic degradation or other processes which result in destruction of, or otherwise elimination or inactivation of, a native T ⁇ RII polypeptide.
  • Such variants, and the genes which encode them can be utilized to alter T ⁇ RII polypeptide levels by modulating the half-life of the T ⁇ RII polypeptides.
  • a short half-life can give rise to more transient biological effects and can allow tighter control of recombinant T ⁇ RII polypeptide levels within the patient.
  • mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
  • a combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential T ⁇ RII polypeptide sequences.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential T ⁇ RII polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • the library of potential T ⁇ RII polypeptide variants can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate vector for expression.
  • the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed.
  • T ⁇ RII polypeptide variants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of T ⁇ RII polypeptides.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Preferred assays include T ⁇ RII ligand binding assays and ligand-mediated cell signaling assays.
  • the T ⁇ RII polypeptides of the disclosure may further comprise post-translational modifications in addition to any that are naturally present in the T ⁇ RII polypeptides.
  • modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation (polyethylene glycol) and acylation.
  • the modified T ⁇ RII polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, mono- or poly-saccharides, and phosphates. Effects of such non-amino acid elements on the functionality of a T ⁇ RII polypeptide may be tested as described herein for other T ⁇ RII polypeptide variants.
  • T ⁇ RII polypeptide When a T ⁇ RII polypeptide is produced in cells by cleaving a nascent form of the T ⁇ RII polypeptide, post-translational processing may also be important for correct folding and/or function of the protein.
  • Different cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK-293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the T ⁇ RII polypeptides.
  • T ⁇ RII polypeptides include fusion proteins having at least a portion of the T ⁇ RII polypeptides and one or more fusion domains.
  • fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.
  • GST glutathione S transferase
  • Fc immunoglobulin heavy chain constant region
  • MBP maltose binding protein
  • human serum albumin human serum albumin.
  • a fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
  • matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used.
  • Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners.
  • a fusion domain may be selected so as to facilitate detection of the T ⁇ RII polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available.
  • fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
  • a T ⁇ RII polypeptide is fused with a domain that stabilizes the T ⁇ RII polypeptide in vivo (a “stabilizer” domain).
  • stabilizing is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains.
  • the present disclosure provides fusion proteins comprising variants of T ⁇ RII polypeptides fused to one of three Fc domain sequences (e.g., SEQ ID NOs: 19, 20, and 21).
  • the Fc domain has one or more mutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered in accordance with the corresponding full-length IgG).
  • the mutant Fc domain having one or more of these mutations e.g., Asp-265 mutation
  • the mutant Fc domain having one or more of these mutations has reduced ability of binding to the Fc ⁇ receptor relative to a wildtype Fc domain.
  • the mutant Fc domain having one or more of these mutations has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fc domain.
  • FcRN MHC class I-related Fc-receptor
  • a T ⁇ RII polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to a T ⁇ RII polypeptide.
  • the T ⁇ RII polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
  • an immunoglobulin Fc domain or simply “Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof.
  • an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region.
  • the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain.
  • the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Ig ⁇ ) ( ⁇ subclasses 1, 2, 3, or 4).
  • IgG immunoglobulin
  • Other classes of immunoglobulin, IgA (Ig ⁇ ), IgD (Ig ⁇ ), IgE (Ig ⁇ ) and IgM (Ig ⁇ ) may be used.
  • the choice of appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087 and 5,726,044.
  • the choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art.
  • the portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CH 3 domain of Fc gamma or the homologous domains in any of IgA, IgD, IgE, or IgM.
  • substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed herein.
  • One example would be to introduce amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for Fc receptors (Cole et al. (1997) J. Immunol. 159:3613).
  • the present disclosure makes available isolated and/or purified forms of the T ⁇ RII polypeptides, which are isolated from, or otherwise substantially free of (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% free of), other proteins and/or other T ⁇ RII polypeptide species.
  • T ⁇ RII polypeptides will generally be produced by expression from recombinant nucleic acids.
  • the disclosure includes nucleic acids encoding soluble T ⁇ RII polypeptides comprising the coding sequence for an extracellular portion of a T ⁇ RII protein.
  • this disclosure also pertains to a host cell comprising such nucleic acids.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a polypeptide of the present disclosure may be expressed in bacterial cells such as E. coli , insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. Accordingly, some embodiments of the present disclosure further pertain to methods of producing the T ⁇ RII polypeptides.
  • the disclosure provides isolated and/or recombinant nucleic acids encoding any of the T ⁇ RII polypeptides, including fragments, functional variants and fusion proteins disclosed herein.
  • SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 encode variants of T ⁇ RII extracellular domain fused to an IgG2 Fc or an N-terminally truncated IgG1 Fc domain.
  • the subject nucleic acids may be single-stranded or double stranded.
  • Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making T ⁇ RII polypeptides or as direct therapeutic agents (e.g., in an antisense, RNAi or gene therapy approach).
  • the subject nucleic acids encoding T ⁇ RII polypeptides are further understood to include nucleic acids that are variants of SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.
  • Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.
  • the disclosure provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.
  • nucleic acid sequences complementary to SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44, and variants of SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 are also within the scope of this disclosure.
  • the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • nucleic acids of the disclosure also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequences designated in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 complement sequences of SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44, or fragments thereof.
  • appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0 ⁇ SSC at 50° C.
  • SSC sodium chloride/sodium citrate
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 ⁇ SSC at 50° C. to a high stringency of about 0.2 ⁇ SSC at 50° C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • the disclosure provides nucleic acids which hybridize under low stringency conditions of 6 ⁇ SSC at room temperature followed by a wash at 2 ⁇ SSC at room temperature.
  • Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 due to degeneracy in the genetic code are also within the scope of the disclosure.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.
  • corresponding variants based on the long isoform of T ⁇ RII will include nucleotide sequences encoding the 25-amino acid insertion along with a conservative Val-Ile substitution at the flanking position C-terminal to the insertion. It will also be appreciated that corresponding variants based on either the long (A) or short (B) isoforms of T ⁇ RII will include variant nucleotide sequences comprising an insertion of 108 nucleotides, encoding a 36-amino-acid insertion (SEQ ID NO: 18), at the same location described for naturally occurring T ⁇ RII isoform C (see Exemplification).
  • the recombinant nucleic acids of the disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression.
  • suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
  • the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a T ⁇ RII polypeptide and operably linked to at least one regulatory sequence.
  • Regulatory sequences are art-recognized and are selected to direct expression of the T ⁇ RII polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology , Academic Press, San Diego, Calif. (1990).
  • any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding a T ⁇ RII polypeptide.
  • useful expression control sequences include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of pro
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
  • a recombinant nucleic acid included in the disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
  • Expression vehicles for production of a recombinant T ⁇ RII polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • bacterial plasmids such as pBR322
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • pREP-derived and p205 Epstein-Barr virus
  • examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems.
  • the various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).
  • pVL-derived vectors such as pVL1392, pVL1393 and pVL941
  • pAcUW-derived vectors such as pAcUW1
  • pBlueBac-derived vectors such as the B-gal containing pBlueBac III.
  • a vector will be designed for production of the subject T ⁇ RII polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wisc.).
  • a vector will be designed for production of the subject T ⁇ RII polypeptides in HEK-293 cells.
  • the subject gene constructs can be used to cause expression of the subject T ⁇ RII polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.
  • This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44) for one or more of the subject T ⁇ RII polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a T ⁇ RII polypeptide disclosed herein may be expressed in bacterial cells such as E. coli , insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
  • the present disclosure further pertains to methods of producing the subject T ⁇ RII polypeptides.
  • a host cell transfected with an expression vector encoding a T ⁇ RII polypeptide can be cultured under appropriate conditions to allow expression of the T ⁇ RII polypeptide to occur.
  • the T ⁇ RII polypeptide may be secreted and isolated from a mixture of cells and medium containing the T ⁇ RII polypeptide.
  • the T ⁇ RII polypeptide may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, and media. Suitable media for cell culture are well known in the art.
  • the subject T ⁇ RII polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of the T ⁇ RII polypeptides and affinity purification with an agent that binds to a domain fused to the T ⁇ RII polypeptide (e.g., a protein A column may be used to purify an T ⁇ RII-Fc fusion).
  • the T ⁇ RII polypeptide is a fusion protein containing a domain which facilitates its purification.
  • purification may be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant T ⁇ RII polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni 2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified T ⁇ RII polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).
  • fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology , eds. Ausubel et al., John Wiley & Sons: 1992).
  • a nucleic acid compound may be single or double stranded.
  • a double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single-stranded.
  • a single-stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called “hairpin” or “stem-loop” structure, with a region of double helical structure.
  • a nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length T ⁇ RII nucleic acid sequence or ligand nucleic acid sequence.
  • the region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, such as between 15 and 25 nucleotides.
  • a region of complementarity may fall within an intron, a coding sequence, or a noncoding sequence of the target transcript, such as the coding sequence portion.
  • a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, such as about 14 to about 50 nucleotides.
  • a nucleic acid may be a DNA (particularly for use as an antisense), RNA, or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a double-stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid).
  • An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds.
  • the strand complementary to the target transcript will generally be RNA or modifications thereof.
  • the other strand may be RNA, DNA, or any other variation.
  • the duplex portion of double-stranded or single-stranded “hairpin” RNAi construct will preferably have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate.
  • Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms.
  • Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90%, or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar. Nucleic acid compounds may also be tested for effects on, for example, angiogenesis.
  • the application further provides T ⁇ RII-Fc fusion proteins with engineered or variant Fc regions.
  • Such antibodies and Fc fusion proteins may be useful, for example, in modulating effector functions, such as, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Additionally, the modifications may improve the stability of the antibodies and Fc fusion proteins.
  • Amino acid sequence variants of the antibodies and Fc fusion proteins are prepared by introducing appropriate nucleotide changes into the DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies and Fc fusion proteins disclosed herein.
  • amino acid changes also may alter post-translational processes of the antibodies and Fc fusion proteins, such as changing the number or position of glycosylation sites.
  • Antibodies and Fc fusion proteins with reduced effector function may be produced by introducing changes in the amino acid sequence, including, but are not limited to, the Ala-Ala mutation described by Bluestone et al. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 Cell Immunol 200; 16-26).
  • antibodies and Fc fusion proteins of the disclosure with mutations within the constant region including the Ala-Ala mutation may be used to reduce or abolish effector function.
  • antibodies and Fc fusion proteins may comprise a mutation to an alanine at position 234 or a mutation to an alanine at position 235, or a combination thereof.
  • the antibody or Fc fusion protein comprises an IgG4 framework, wherein the Ala-Ala mutation would describe a mutation(s) from phenylalanine to alanine at position 234 and/or a mutation from leucine to alanine at position 235.
  • the antibody or Fc fusion protein comprises an IgG1 framework, wherein the Ala-Ala mutation would describe a mutation(s) from leucine to alanine at position 234 and/or a mutation from leucine to alanine at position 235.
  • the antibody or Fc fusion protein may alternatively or additionally carry other mutations, including the point mutation K322A in the CH2 domain (Hezareh et al. 2001 J. Virol. 75: 12161-8).
  • the antibody or Fc fusion protein may be modified to either enhance or inhibit complement dependent cytotoxicity (CDC).
  • Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region (see, e.g., U.S. Pat. No. 6,194,551).
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved or reduced internalization capability and/or increased or decreased complement-mediated cell killing. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.
  • the present disclosure relates in part to the discovery that the TGF ⁇ type II receptor (T ⁇ RII) binds to GDF15 with high affinity.
  • T ⁇ RII TGF ⁇ type II receptor
  • GDF15 has not been shown biochemically to bind or interact directly with a receptor. Inadequate or inappropriate ligand purification could be a potential reason for the inactivity of commercially available GDF15.
  • Exemplary GDF15 polypeptides demonstrating a T ⁇ RII binding activity and methods of making and purifying such polypeptides are disclosed herein. Sequences of native precursor GDF15 proteins and nucleotides from human and mouse are set forth in FIGS. 1-4 . Mature human GDF15 extends from residues 197 to 308 of SEQ ID NO: 1.
  • mature mouse GDF15 extends from residues 192 to 303 of SEQ ID NO: 3.
  • the present disclosure makes available isolated and/or purified forms of the GDF15 polypeptides or fragments thereof, which are isolated from, or otherwise substantially free of (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% free of), other proteins and/or other GDF15 polypeptide species.
  • the GDF15 polypeptides of the disclosure bind to T ⁇ RII with high affinity. Binding may be assessed using purified proteins in solution or in a surface plasmon resonance system, such as a BiacoreTM system.
  • the GDF15 polypeptides will have an affinity (a dissociation constant) of about 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 M or less for T ⁇ RII polypeptides.
  • affinity a dissociation constant
  • the GDF15 polypeptides of the disclosure are isolated and purified according to methods described herein.
  • GDF15 polypeptides will generally be produced by expression from recombinant nucleic acids.
  • GDF15 polypeptides may include a polypeptide consisting of, or comprising, an amino acid sequence at least 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the GDF15 polypeptide of SEQ ID NO: 1 or SEQ ID NO: 3, or a functional fragment thereof
  • GDF15 polypeptides may include a polypeptide consisting of, or comprising, an amino acid sequence at least 80% identical, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the GDF15 polypeptide comprising residues 197 to 308 of SEQ ID NO: 1 or residues 192 to 303 of SEQ ID NO: 3, or a functional fragment thereof.
  • the unprocessed GDF15 polypeptide may either include or exclude any signal sequence, as well as any sequence N-terminal to the signal sequence.
  • a GDF15 polypeptide may include variants of SEQ ID NO: 1 or SEQ ID NO: 3, or portions thereof, corresponding to 197 to 308 of SEQ ID NO: 1 or residues 192 to 303 of SEQ ID NO: 3, respectively (including variants that comprise, for example, no more than 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acid substitutions in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3), fragments thereof, and fusion proteins comprising any of the foregoing, but in each case preferably any of the foregoing GDF15 polypeptides will possess substantial affinity for a T ⁇ RII polypeptide.
  • the GDF15 polypeptides of the disclosure may further comprise post-translational modifications in addition to any that are naturally present in the GDF15 polypeptides.
  • modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation (polyethylene glycol) and acylation.
  • the modified GDF15 polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, mono- or poly-saccharides, and phosphates. Effects of such non-amino acid elements on the functionality of a GDF15 polypeptide may be tested as described herein for other GDF15 polypeptides.
  • a GDF15 polypeptide When a GDF15 polypeptide is produced in cells by cleaving a nascent form of the GDF15 polypeptide, post-translational processing may also be important for correct folding and/or function of the protein.
  • Different cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK-293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the GDF15 polypeptides.
  • the disclosure includes nucleic acids encoding precursor and mature GDF15 polypeptides.
  • this disclosure also pertains to a host cell comprising such nucleic acids.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a polypeptide of the present disclosure may be expressed in bacterial cells such as E. coli , insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. Accordingly, some embodiments of the present disclosure further pertain to methods of producing the GDF15 polypeptides.
  • the disclosure provides isolated and/or recombinant nucleic acids encoding any of the GDF15 polypeptides, including fragments, functional variants and fusion proteins disclosed herein.
  • the subject nucleic acids may be single-stranded or double-stranded.
  • Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making GDF15 polypeptides or as direct therapeutic agents (e.g., in an antisense, RNAi or gene therapy approach).
  • the subject nucleic acids encoding GDF15 polypeptides are further understood to include nucleic acids that are variants of SEQ ID NO: 1 or SEQ ID NO: 3.
  • Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.
  • the disclosure provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.
  • nucleic acid sequences complementary to SEQ ID NO: 1 or SEQ ID NO: 3 and variants of SEQ ID NO: 1 or SEQ ID NO: 3 are also within the scope of this disclosure.
  • the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • nucleic acids of the disclosure also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequences designated in SEQ ID NO: 1 or SEQ ID NO: 3, complement sequences of SEQ ID NO: 1 or SEQ ID NO: 3, or fragments thereof.
  • appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0 ⁇ SSC at 50° C.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 ⁇ SSC at 50° C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • the disclosure provides nucleic acids which hybridize under low stringency conditions of 6 ⁇ SSC at room temperature followed by a wash at 2 ⁇ SSC at room temperature.
  • Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 due to degeneracy in the genetic code are also within the scope of the disclosure.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.
  • the recombinant nucleic acids of the disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression.
  • suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
  • the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a GDF15 polypeptide and operably linked to at least one regulatory sequence.
  • Regulatory sequences are art-recognized and are selected to direct expression of the GDF15 polypeptide.
  • the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology , Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding a GDF15 polypeptide.
  • Such useful expression control sequences include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
  • a recombinant nucleic acid included in the disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
  • Expression vehicles for production of a recombinant GDF15 polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • bacterial plasmids such as pBR322
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • pREP-derived and p205 Epstein-Barr virus
  • examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems.
  • the various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).
  • pVL-derived vectors such as pVL1392, pVL1393 and pVL941
  • pAcUW-derived vectors such as pAcUW1
  • pBlueBac-derived vectors such as the B-gal containing pBlueBac III.
  • a vector will be designed for production of the subject T ⁇ RII polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.), pCI-neo vectors (Promega, Madison, Wisc.) and UCOETM-derived vectors (Millipore).
  • a Pcmv-Script vector Stratagene, La Jolla, Calif.
  • pcDNA4 vectors Invitrogen, Carlsbad, Calif.
  • pCI-neo vectors Promega, Madison, Wisc.
  • UCOETM-derived vectors UCOETM-derived vectors
  • This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NO: 1 or SEQ ID NO:3) for one or more of the subject GDF15 polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a GDF15 polypeptide disclosed herein may be expressed in bacterial cells such as E. coli , insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
  • a GDF15 polypeptide disclosed herein is expressed in CHO cells.
  • the present disclosure further pertains to methods of producing the subject GDF15 polypeptides.
  • a host cell transfected with an expression vector encoding a GDF15 polypeptide can be cultured under appropriate conditions to allow expression of the GDF15 polypeptide to occur.
  • the GDF15 polypeptide may be secreted and isolated from a mixture of cells and medium containing the GDF15 polypeptide.
  • the T ⁇ RII polypeptide may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells and media. Suitable media for cell culture are well known in the art.
  • the subject GDF15 polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of the GDF15 polypeptides and affinity purification with an agent that binds to a domain fused to the GDF15 polypeptide (e.g., a protein A column may be used to purify an GDF15-Fc fusion).
  • the GDF15 polypeptide is a fusion protein containing a domain which facilitates its purification.
  • purification may be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • the subject GDF15 polypeptides are purified from culture media using a series of cation-exchange column chromatography steps.
  • the material used for the cation exchange column can be resins having substituents such as carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).
  • Examples of the material used for the cation exchange column chromatography include SP SepharoseTM Fast Flow, Q SepharoseTM Fast Flow, DEAE SepharoseTM Fast Flow, CaptoTM S, CaptoTM DEAE (GE Healthcare), S HyperCelTM (Pall), TOYOPEARL GigaCap S-650 (TOSOH) or weak cation exchangers such as carboxymethyl.
  • SP SepharoseTM Fast Flow and Q SepharoseTM Fast Flow are preferred.
  • parameters of the conditioned media from host cells stably expressing a GDF15 polypeptide such as pH, ionic strength, and temperature may be adjusted if necessary.
  • a chromatography column is flushed and equilibrated with one or more solutions prior to contact with a polypeptide containing supernatant.
  • Such solutions can include, for example, a buffer (e.g., Tris, MES, HEPES, histidine, phosphate or sodium acetate, e.g., between 1-500 mM, 25-100 mM, 15-30 mM or 20 mM), and/or salt (e.g., NaCl, NaPO 4 sodium acetate, or CaCl 2 , e.g., between 0-2 M, 1-2 M or 500 mM-1M).
  • the pH of an equilibration solution generally ranges from 3.5-10 (e.g., between pH 3.5-6, 4.0-5.5, 4.5-4.8 or 4.7).
  • Wash solutions can include a buffer (e.g., Tris, MES, HEPES, histidine, phosphate, or sodium acetate, e.g., between 1-500 mM, 25-100 mM, 15-30 mM or 20 mM), and/or salt (e.g., NaCl, NaPO 4 , sodium acetate, or CaCl 2 , e.g., between 0-2 M, 1-2 M, 100 mM-1M or 100 mM-500 mM), and/or an additive (e.g.
  • a buffer e.g., Tris, MES, HEPES, histidine, phosphate, or sodium acetate, e.g., between 1-500 mM, 25-100 mM, 15-30 mM or 20 mM
  • salt e.g., NaCl, NaPO 4 , sodium acetate, or CaCl 2 , e.g., between 0-2 M, 1-2 M, 100
  • wash solutions generally have a pH between 3.5 and 10 (e.g., a pH between 4.5-8.0).
  • Polypeptides can be eluted from a column using a step or gradient change in pH, salt type, salt concentration, solvent type, solvent concentration, displacer type, displacer concentration, or a combination thereof. In general, to elute a polypeptide from a column, the medium is contacted with an elution buffer.
  • an elution buffer elution buffer contains a buffer (e.g., HEPES or Tris, e.g., 10-100 mM, 25-75 mM or 50 mM) and/or contains a salt (e.g., NaCl or CaCl 2 , e.g., 0-2 M, e.g., 10-100 mM).
  • a buffer e.g., HEPES or Tris, e.g., 10-100 mM, 25-75 mM or 50 mM
  • a salt e.g., NaCl or CaCl 2 , e.g., 0-2 M, e.g., 10-100 mM.
  • an elution buffer may contain glycine, acetic acid, or citric acid (e.g., 20-250 mM, or 150 mM).
  • An elution buffer may also contain acetic acid (e.g., 20 mM to about 50
  • the pH of the elution buffer may range from about 5.0 to about 10.0. In some embodiments, pH can be changed (e.g., gradually) to produce a gradient elution. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, a series of column chromatography steps are performed.
  • T ⁇ RII polypeptides act as antagonists of GDF15 signaling.
  • soluble T ⁇ RII polypeptides, and particularly T ⁇ RII-Fc are preferred antagonists, other types of GDF15 antagonists are expected to be useful, including anti-GDF15 antibodies, anti-T ⁇ RII antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit the production of GDF15 or T ⁇ RII and other inhibitors of GDF15 or T ⁇ RII, particularly those that disrupt GDF15-T ⁇ RII binding.
  • an antibody that is specifically reactive with a GDF15 polypeptide and which either binds to GDF15 polypeptide so as to compete with its binding to T ⁇ RII polypeptide (binding competitively) or otherwise inhibits GDF15-mediated signaling may be used as an antagonist of GDF15 polypeptide activities.
  • an antibody that is specifically reactive with a T ⁇ RII polypeptide and which disrupts GDF15 binding may be used as an antagonist.
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the GDF15 polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of a GDF15 or T ⁇ RII polypeptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antibody-producing cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a GDF15 polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with a subject polypeptide. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • the antibody of the present invention is further intended to include bispecific, single-chain, chimeric, humanized and fully human molecules having affinity for an T ⁇ RII or GDF15 polypeptide conferred by at least one CDR region of the antibody.
  • An antibody may further comprise a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).
  • the antibody is a recombinant antibody, which term encompasses any antibody generated in part by techniques of molecular biology, including CDR-grafted or chimeric antibodies, human or other antibodies assembled from library-selected antibody domains, single chain antibodies and single domain antibodies (e.g., human V H proteins or camelid V HH proteins).
  • an antibody of the invention is a monoclonal antibody, and in certain embodiments, the invention makes available methods for generating novel antibodies.
  • a method for generating a monoclonal antibody that binds specifically to a GDF15 polypeptide or T ⁇ RII polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monoclonal antibody that binds specifically to the antigen.
  • antibody-producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the antigen.
  • the monoclonal antibody may be purified from the cell culture.
  • the adjective “specifically reactive with” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., a GDF15 polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross-reacting polypeptides. One characteristic that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen.
  • antibodies will have an affinity (a dissociation constant) of about 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 or less.
  • affinity a dissociation constant
  • a neutralizing anti-GDF15 or anti-T ⁇ RII antibody would generally have a dissociation constant of 10 ⁇ 9 or less.
  • the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding.
  • a variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Md.), western blots, immunoprecipitation assays, and immunohistochemistry.
  • nucleic acid compounds examples include antisense nucleic acids, RNAi constructs and catalytic nucleic acid constructs.
  • a nucleic acid compound may be single- or double-stranded.
  • a double-stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single-stranded.
  • a single-stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called “hairpin” or “stem-loop” structure, with a region of double helical structure.
  • a nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length GDF15 nucleic acid sequence or T ⁇ RII nucleic acid sequence.
  • the region of complementarity will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, such as between 15 and 25 nucleotides.
  • a region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as the coding sequence portion.
  • a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, such as about 14 to about 50 nucleotides.
  • a nucleic acid may be a DNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a double-stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid).
  • An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds.
  • the strand complementary to the target transcript will generally be RNA or modifications thereof.
  • the other strand may be RNA, DNA or any other variation.
  • the duplex portion of double-stranded or single-stranded “hairpin” RNAi construct will preferably have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate.
  • Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms.
  • Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90% or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar.
  • the present invention relates to the use of T ⁇ RII polypeptides (e.g., soluble T ⁇ RII polypeptides) and GDF15 polypeptides to identify compounds (agents) which are agonist or antagonists of the GDF15-T ⁇ RII signaling pathway.
  • T ⁇ RII polypeptides e.g., soluble T ⁇ RII polypeptides
  • GDF15 polypeptides e.g., soluble T ⁇ RII polypeptides
  • Compounds identified through this screening can be tested to assess their ability to modulate GDF15 signaling activity in vitro.
  • these compounds can further be tested in animal models to assess their ability to modulate tissue growth in vivo.
  • high-throughput screening of compounds can be carried out to identify agents that perturb GDF15 or T ⁇ RII-mediated cell signaling.
  • the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of a T ⁇ RII polypeptide to GDF15.
  • the assay can be used to identify compounds that enhance binding of a T ⁇ RII polypeptide to GDF15.
  • the compounds can be identified by their ability to interact with a GDF15 or T ⁇ RII polypeptide.
  • test compounds (agents) of the invention may be created by any combinatorial chemical method.
  • the subject compounds may be naturally occurring biomolecules synthesized in vivo or in vitro.
  • Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly.
  • Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules.
  • the test agent is a small organic molecule having a molecular weight of less than about 2,000 daltons.
  • test compounds of the invention can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry.
  • libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds.
  • Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.
  • the compounds may be optionally derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds.
  • Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivatible crosslinkers or any combinations thereof.
  • the compound of interest is contacted with an isolated and purified T ⁇ RII polypeptide which is ordinarily capable of binding to GDF15.
  • a composition containing a T ⁇ RII ligand is then added to the mixture of the compound and T ⁇ RII polypeptide.
  • Detection and quantification of T ⁇ RII/GDF15 complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the T ⁇ RII polypeptide and GDF15.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • T ⁇ RII/GDF15 for example, in a control assay, isolated and a purified GDF15 is added to a composition containing the T ⁇ RII polypeptide, and the formation of T ⁇ RII/GDF15 complex is quantitated in the absence of the test compound.
  • reactants may be admixed can be varied, and can be admixed simultaneously.
  • cellular extracts and lysates may be used to render a suitable cell-free assay system.
  • Complex formation between the T ⁇ RII polypeptide and GDF15 may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (e.g., 32 P, 355 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzymatically labeled T ⁇ RII polypeptide or GDF15, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled (e.g., 32 P, 355 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzymatically labeled T ⁇ RII polypeptide or GDF15, by immunoassay, or by chromatographic detection.
  • the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between a T ⁇ RII polypeptide and its binding protein.
  • FRET fluorescence resonance energy transfer
  • other modes of detection such as those based on optical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the invention.
  • the present invention contemplates the use of an interaction trap assay, also known as the “two hybrid assay,” for identifying agents that disrupt or potentiate interaction between a T ⁇ RII polypeptide and its binding protein.
  • an interaction trap assay also known as the “two hybrid assay” for identifying agents that disrupt or potentiate interaction between a T ⁇ RII polypeptide and its binding protein. See for example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696).
  • the present invention contemplates the use of reverse two hybrid systems to identify compounds (e.g., small molecules or peptides) that dissociate interactions between a T ⁇ RII polypeptide and its binding protein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; and U.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.
  • compounds e.g., small molecules or peptides
  • the subject compounds are identified by their ability to interact with a T ⁇ RII or GDF15 polypeptide of the invention.
  • the interaction between the compound and the T ⁇ RII or GDF15 polypeptide may be covalent or non-covalent.
  • such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).
  • the compounds may be screened in a mechanism based assay, such as an assay to detect compounds which bind to a GDF15 or T ⁇ RII polypeptide. This may include a solid-phase or fluid-phase binding event.
  • the gene encoding a GDF15 or T ⁇ RII polypeptide can be transfected with a reporter system (e.g., ⁇ -galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library preferably by a high-throughput screening or with individual members of the library.
  • a reporter system e.g., ⁇ -galactosidase, luciferase, or green fluorescent protein
  • Other mechanism-based binding assays may be used, for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound compounds may be detected usually using colorimetric or fluorescence or surface plasmon resonance.
  • the present invention provides methods and agents for modulating (stimulating or inhibiting) GDF15-mediated cell signaling. Therefore, any compound identified can be tested in whole cells or tissues, in vitro or in vivo, to confirm their ability to modulate GDF15 signaling. Various methods known in the art can be utilized for this purpose.
  • a therapeutic that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • treating includes amelioration or elimination of the condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician and the intended result of administration of the therapeutic agent.
  • the disclosure provides methods of treating or preventing a disease or condition associated with a TGF ⁇ superfamily member by administering to a subject an effective amount of a T ⁇ RII polypeptide, including a T ⁇ RII-Fc fusion protein or nucleic acid antagonists (e.g., antisense or siRNA) of the foregoing, hereafter collectively referred to as “therapeutic agents”.
  • a T ⁇ RII polypeptide including a T ⁇ RII-Fc fusion protein or nucleic acid antagonists (e.g., antisense or siRNA) of the foregoing, hereafter collectively referred to as “therapeutic agents”.
  • the disease or condition is associated with dysregulated GDF15, TGF ⁇ 1 or TGF ⁇ 3 signaling.
  • methods and compositions for treating certain cardiovascular or vascular disorders are also provided.
  • the disclosure provides methods and compositions for treating or preventing cancer.
  • the disclosure provides methods and compositions for treating or preventing fibrotic disorders and conditions.
  • polypeptide therapeutic agents of the present disclosure are useful for treating or preventing chronic vascular or cardiovascular diseases.
  • exemplary disorders of this kind include, but are not limited to, heart disease (including myocardial disease, myocardial infarct, angina pectoris, and heart valve disease); renal disease (including chronic glomerular inflammation, diabetic renal failure, and lupus-related renal inflammation); disorders associated with atherosclerosis or other types of arteriosclerosis (including stroke, cerebral hemorrhage, subarachnoid hemorrhage, angina pectoris, and renal arteriosclerosis); thrombotic disorders (including cerebral thrombosis, thrombotic intestinal necrosis); complications of diabetes (including diabetes-related retinal disease, cataracts, diabetes-related renal disease, diabetes-related neuropathology, diabetes-related gangrene, and diabetes-related chronic infection); vascular inflammatory disorders (systemic lupus erythematosus, joint rheumatism, joint arterial inflammation, large-cell arterial inflammation, Kawasaki disease, Takay
  • Exemplary disorders further include, but are not limited to, hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, pre-eclampsia, and restenosis.
  • HHT hereditary hemorrhagic telangiectasia
  • Marfan syndrome Marfan syndrome
  • Loeys-Dietz syndrome familial thoracic aortic aneurysm syndrome
  • arterial tortuosity syndrome pre-eclampsia
  • restenosis pre-eclampsia
  • the T ⁇ RII polypeptide can be administered to the subject alone, or in combination with one or more agents or therapeutic modalities, e.g., therapeutic agents, which are useful for treating TGF ⁇ associated cardiovascular disorders and/or conditions.
  • the second agent or therapeutic modality is chosen from one or more of: angioplasty, beta blockers, anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators, hormone antagonists, endothelin antagonists, calcium channel blockers, phosphodiesterase inhibitors, angiotensin type 2 antagonists and/or cytokine blockers/inhibitors
  • polypeptide therapeutic agents of the present disclosure are useful for treating or preventing a cancer (tumor).
  • cancer and “cancerous” refer to or describe, the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • cancer, or neoplastic disorders include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, stomach cancer, intestinal cancer, skin cancer, bone cancer, gastric cancer, melanoma, and various types of head and neck cancer, including squamous cell head and neck cancer.
  • neoplastic disorders and related conditions include esophageal carcinomas, thecomas, arrhenoblastomas, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, and Meigs'
  • a cancer that is particularly amenable to treatment with the therapeutic agents described herein may be characterized by one or more of the following: the cancer has elevated T ⁇ RII levels detectable in the tumor or the serum, increased GDF15, TGF ⁇ 1 or TGF ⁇ 3 expression levels or biological activity, is metastatic or at risk of becoming metastatic, or any combination thereof.
  • polypeptide therapeutic agents can be administered, together (simultaneously) or at different times (sequentially).
  • polypeptide therapeutic agents can be administered with another type of compounds for treating cancer or for inhibiting angiogenesis.
  • the subject methods of the disclosure can be used alone.
  • the subject methods may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor).
  • proliferative disorders e.g., tumor
  • such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy.
  • conventional cancer therapies e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery
  • a wide array of conventional compounds have been shown to have anti-neoplastic or anti-cancer activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies.
  • chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.
  • a therapeutic agent disclosed herein When a therapeutic agent disclosed herein is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such therapeutic agent may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti-neoplastic agent in resistant cells.
  • the polypeptide therapeutic agents described herein may be used in combination with other compositions and procedures for the treatment of diseases.
  • a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with the T ⁇ RII polypeptide, and then the T ⁇ RII polypeptide may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
  • other therapeutic agents useful for combination tumor therapy with a T ⁇ RII polypeptide include other cancer therapies: e.g., surgery, cytotoxic agents, radiological treatments involving irradiation or administration of radioactive substances, chemotherapeutic agents, anti-hormonal agents, growth inhibitory agents, anti-neoplastic compositions, and treatment with anti-cancer agents listed herein and known in the art, or combinations thereof.
  • cancer therapies e.g., surgery, cytotoxic agents, radiological treatments involving irradiation or administration of radioactive substances, chemotherapeutic agents, anti-hormonal agents, growth inhibitory agents, anti-neoplastic compositions, and treatment with anti-cancer agents listed herein and known in the art, or combinations thereof.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g.
  • methotrexate adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.
  • a tumoricidal agent causes destruction of tumor cells.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topote
  • dynemicin including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,
  • anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves.
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX® tamoxifen
  • EVISTA® raloxifene droloxifene
  • 4-hydroxytamoxifen trioxifene, keoxifene, LY1 17018, onapristone, and FARESTON® toremifene
  • anti-progesterones anti-progesterones
  • estrogen receptor down-regulators ETDs
  • agents that function to suppress or shut down the ovaries for example, luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and trip
  • LHRH luteinizing hormone
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROC AL® etidronate, NE-58095, ZOMET A® zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell either in vitro or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • T ⁇ RII polypeptides may be useful in the treatment or prevention of fibrosis.
  • fibrosis refers to the aberrant formation or development of excess fibrous connective tissue by cells in an organ or tissue.
  • processes related to fibrosis can occur as part of normal tissue formation or repair, dysregulation of these processes can lead to altered cellular composition and excess connective tissue deposition that progressively impairs to tissue or organ function.
  • the formation of fibrous tissue can result from a reparative or reactive process.
  • Fibrotic disorders or conditions include, but are not limited to, fibroproliferative disorders associated with vascular diseases, such as cardiac disease, cerebral disease, and peripheral vascular disease, as well as tissues and organ systems including the heart, skin, kidney, peritoneum, gut, and liver (as disclosed in, e.g., Wynn, 2004, Nat Rev 4:583-594, incorporated herein by reference).
  • vascular diseases such as cardiac disease, cerebral disease, and peripheral vascular disease
  • tissues and organ systems including the heart, skin, kidney, peritoneum, gut, and liver (as disclosed in, e.g., Wynn, 2004, Nat Rev 4:583-594, incorporated herein by reference).
  • Exemplary disorders that can be treated include, but are not limited to, renal fibrosis, including nephropathies associated with injury/fibrosis, e.g., chronic nephropathies associated with diabetes (e.g., diabetic nephropathy), lupus, scleroderma, glomerular nephritis, focal segmental glomerular sclerosis, and IgA nephropathy; gut fibrosis, e.g., scleroderma, and radiation-induced gut fibrosis; liver fibrosis, e.g., cirrhosis, alcohol-induced liver fibrosis, biliary duct injury, primary biliary cirrhosis, infection or viral-induced liver fibrosis, congenital hepatic fibrosis and autoimmune hepatitis; and other fibrotic conditions, such as cystic fibrosis, endomyocardial fibrosis, mediastinal fibrosis, sarc
  • fibrotic disorder As used herein, the terms “fibrotic disorder”, “fibrotic condition,” and “fibrotic disease,” are used interchangeably to refer to a disorder, condition or disease characterized by fibrosis.
  • fibrotic disorders include, but are not limited to sclerotic disorders (e.g., scleroderma, atherosclerosis, diffuse systemic sclerosis), vascular fibrosis, pancreatic fibrosis, liver fibrosis (e.g., cirrhosis), renal fibrosis, musculoskeletal fibrosis, cardiac fibrosis (e.g., endomyocardial fibrosis, idiopathic myocardiopathy), skin fibrosis (e.g., scleroderma, post-traumatic, operative cutaneous scarring, keloids and cutaneous keloid formation), eye fibrosis (e.g., glaucoma, sclerosis of the eyes, conjunctival and corneal scarring, and pteryg
  • inhibition of the fibrotic response of a cell includes, but is not limited to the inhibition of the fibrotic response of one or more cells within the liver (or liver tissue); one or more cells within the kidney (or renal tissue); one or more cells within muscle tissue; one or more cells within the heart (or cardiac tissue); one or more cells within the pancreas; one or more cells within the skin; one or more cells within the bone, one or more cells within the vasculature, one or more stem cells, or one or more cells within the eye.
  • the present invention contemplates the use of T ⁇ RII polypeptides in combination with one or more other therapeutic modalities.
  • T ⁇ RII polypeptides in addition to the use of T ⁇ RII polypeptides, one may also administer to the subject one or more “standard” therapies for treating fibrotic disorders.
  • the T ⁇ RII polypeptides can be administered in combination with (i.e., together with) cytotoxins, immunosuppressive agents, radiotoxic agents, and/or therapeutic antibodies.
  • Particular co-therapeutics contemplated by the present invention include, but are not limited to, steroids (e.g., corticosteroids, such as Prednisone), immune-suppressing and/or anti-inflammatory agents (e.g., gamma-interferon, cyclophosphamide, azathioprine, methotrexate, penicillamine, cyclosporine, colchicine, antithymocyte globulin, mycophenolate mofetil, and hydroxychloroquine), cytotoxic drugs, calcium channel blockers (e.g., nifedipine), angiotensin converting enzyme inhibitors (ACE) inhibitors, para-aminobenzoic acid (PABA), dimethyl sulfoxide, transforming growth factor beta (TGF ⁇ ) inhibitors, interleukin-5 (IL-5) inhibitors, and pan caspase inhibitors.
  • steroids e.g., corticosteroids, such as Prednisone
  • Additional anti-fibrotic agents that may be used in combination with T ⁇ RII polypeptides include, but are not limited to, lectins (as described in, for example, U.S. Pat. No. 7,026,283, the entire contents of which is incorporated herein by reference), as well as the anti-fibrotic agents described by Wynn et al (2007, J Clin Invest 117:524-529, the entire contents of which is incorporated herein by reference).
  • additional anti-fibrotic agents and therapies include, but are not limited to, various anti-inflammatory/immunosuppressive/cytotoxic drugs (including colchicine, azathioprine, cyclophosphamide, prednisone, thalidomide, pentoxifylline and theophylline), TGF ⁇ signaling modifiers (including relaxin, SMAD7, HGF, and BMP7, as well as TGF ⁇ 1, T ⁇ RI, T ⁇ RII, EGR-I, and CTGF inhibitors), cytokine and cytokine receptor antagonists (inhibitors of IL-1 ⁇ , IL-5, IL-6, IL-13, IL-21, IL-4R, IL-13R ⁇ 1, GM-CSF, TNF- ⁇ , oncostatin M, WlSP-I, and PDGFs), cytokines and chemokincs (IFN- ⁇ , IFN- ⁇ / ⁇ , IL-12, IL-10, HGF, CXCL10, and CX
  • the T ⁇ RII polypeptide and the co-therapeutic agent or co-therapy can be administered in the same formulation or separately.
  • the T ⁇ RII polypeptide can be administered before, after, or concurrently with the co-therapeutic or co-therapy.
  • One agent may precede or follow administration of the other agent by intervals ranging from minutes to weeks.
  • two or more different kinds of therapeutic agents are applied separately to a subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that these different kinds of agents would still be able to exert an advantageously combined effect on the target tissues or cells.
  • compositions for use in accordance with the present disclosure may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Such formulations will generally be substantially pyrogen-free, in compliance with most regulatory requirements.
  • the therapeutic method of the disclosure includes administering the composition systemically, or locally as an implant or device.
  • the therapeutic composition for use in this disclosure is in a pyrogen-free, physiologically acceptable form.
  • Therapeutically useful agents other than the T ⁇ RII signaling antagonists which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds (e.g., T ⁇ RII polypeptides) in the methods disclosed herein.
  • compositions suitable for parenteral administration may comprise one or more T ⁇ RII polypeptides in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions and formulations may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration
  • compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., T ⁇ RII polypeptides) to a target tissue site, providing a structure for the developing tissue and optimally capable of being resorbed into the body.
  • the matrix may provide slow release of the T ⁇ RII polypeptides.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.
  • the particular application of the subject compositions will define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • methods of the invention can be administered for orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient.
  • An agent may also be administered as a bolus, electuary or paste.
  • one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents,
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the subject compounds of the invention (e.g., T ⁇ RII polypeptides).
  • the various factors include, but are not limited to, the patient's age, sex, and diet, the severity disease, time of administration, and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and the types of compounds in the composition.
  • the addition of other known growth factors to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of bone growth and/or repair, for example, X-rays (including DEXA), histomorphometric determinations, and tetracycline labeling.
  • the present invention also provides gene therapy for the in vivo production of T ⁇ RII polypeptides.
  • Such therapy would achieve its therapeutic effect by introduction of the T ⁇ RII polynucleotide sequences into cells or tissues having the disorders as listed above.
  • Delivery of T ⁇ RII polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • Preferred for therapeutic delivery of T ⁇ RII polynucleotide sequences is the use of targeted liposomes.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • Retroviral vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody.
  • specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the T ⁇ RII polynucleotide.
  • the vector is targeted to bone or cartilage.
  • tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
  • compositions of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
  • the targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art.
  • the disclosure provides formulations that may be varied to include acids and bases to adjust the pH; and buffering agents to keep the pH within a narrow range.
  • GDF15 also known as macrophage-inhibitory cytokine-1
  • GDF15 has not been shown biochemically to bind or interact directly with any receptor. Applicants first tried without success to identify a native receptor with high-affinity binding to GDF15 using commercially available human GDF15 (R&D Systems) produced in mammalian CHO cells.
  • mature GDF15 is synthesized with a larger prodomain (Harrison et al., Growth Factors 29:174, 2011; Shi et al., Nature 474:343, 2011) that is removed through cleavage by a furin-like protease at the canonical RXXR site to generate mature dimeric GDF15. Since inadequate or inappropriate ligand purification could be a potential reason for inactivity of commercially available GDF15, Applicants tested different purification procedures for GDF15.
  • CHO cells to express human GDF15 (hGDF15) and murine GDF15 (mGDF15) for further studies.
  • the amino acid sequence of native precursor for hGDF15 is shown in FIG. 1
  • a corresponding nucleotide sequence (with a silent, single nucleotide substitution compared to the native sequence) is shown in FIG. 2 .
  • the native amino acid and nucleotide sequences for mGDF15 precursor are shown in FIGS. 3 and 4 , respectively.
  • UCOETM-based constructs encoding human or murine GDF15 precursor were stably transfected into a CHO-PACE cell line.
  • Clones were selected in methotrexate levels of 10 nM, 20 nM, and 50 nM, and any clones that formed colonies (one or two per methotrexate concentration) were then pooled. No gene amplification was performed since it is difficult to amplify UCOETM pools while maintaining stability of expression. Instead of dilution cloning, high-expressing pools were identified and used for generating hGDF15 and mGDF15.
  • conditioned media from CHO cells stably expressing hGDF15 was adjusted to pH 4.7 with acetic acid. After incubation of media for 10 min at ambient temperature, precipitate was removed by centrifugation. Supernatant was filtered with a 0.8 ⁇ m disposable filter.
  • An SP SepharoseTM Fast Flow column (GE Healthcare) was equilibrated with buffers A (20 mM sodium acetate, pH 4.7) and B (20 mM sodium acetate, 1M NaCl, pH 4.7). Loading was performed at 100 cm/hr. The column was washed with 20% B (200 mM NaCl) until no more protein eluted from the column and then washed back to 0% B to remove any residual salt.
  • Protein was eluted with 50 mM Tris, 6M urea, pH 8.0 (Tris+urea pool) until no more protein eluted from the column, followed by elution with 50 mM Tris, 6M urea, 1M NaCl, pH 8.0 (Tris+urea+salt pool). Each pool was dialyzed in 50 mM 4-morpholineethanesulfonic acid (MES, pH 6.5) overnight at 4° C.
  • MES 4-morpholineethanesulfonic acid
  • GDF15 found in the Tris+urea+salt pool was degraded based on Western blot analysis, so this pool was discarded.
  • the Tris+urea pool was loaded on a Q SepharoseTM Fast Flow column (GE Healthcare) previously equilibrated with buffers A (50 mM MES, pH 6.5) and B (50 mM MES, 1M NaCl, pH 6.5). The flow-through was collected, and the column was washed with 10% B (100 mM NaCl), followed by a 10-50% B gradient (100-500 mM NaCl) over five column volumes at 120 cm/hr. After evaluation of the flow-through and wash fractions by Western blot, protein was found mainly in the flow-through.
  • the flow-through was injected on a reverse-phase preparative C4 column (Vydac) attached to a HPLC, with buffers A (water/0.1% TFA) and B (acetonitrile/0.1% TFA).
  • a 25-40% B gradient over 1 h at 4.5 mL/min produced the best resolution.
  • Collected fractions were evaluated by SDS-PAGE gel (Sypro Ruby) and Western blot to select those for concentration in a centrifugal evaporator.
  • the pH of the conditioned media was adjusted to pH 4.7 with acetic acid. After incubation of media for 10 min at ambient temperature, precipitate was removed by centrifugation. Supernatant was filtered with a 0.8 ⁇ m disposable filter. An SP SepharoseTM Fast Flow column (GE Healthcare) was equilibrated with buffers A (20 mM sodium acetate, pH 4.7) and B (20 mM sodium acetate, 1M NaCl, pH 4.7). Loading was performed at 100-150 cm/hr, and the column was washed with buffer A until no more protein eluted from the column.
  • a wash was performed at 60% B (600 mM NaCl) for 3-4 column volumes, followed by elution with 100% B (1M NaCl) for 3-4 column volumes. Elution continued with 50 mM Tris, 6M urea, pH 8.0, to remove any protein still bound to the resin.
  • Non-reduced samples of SP-column fractions were analyzed by Western blot. Although most protein was found in the Tris-eluted fractions, previous experiments have indicated that mGDF15 found in these fractions is essentially inactive, so it was not used for further purification. Instead, purification was continued with protein found in the 100% B elution (salt-elution pool). This pool was injected on a reverse-phase preparative C4 column (Vydac) attached to a HPLC. Buffer A was water/0.1% TFA and buffer B was acetonitrile/0.1% TFA. Protein was eluted with a 25-40% B gradient over 1 h at 4.5 mL/min. After evaluation of the reverse-phase column fractions by SDS-PAGE gel (Sypro Ruby) and Western blot, the fractions containing pure mGDF15 were pooled and concentrated in a centrifugal evaporator.
  • hGDF15 and mGDF15 were each confirmed by N-terminal sequencing. Both types of purified GDF15 stimulated SMAD2/3 phosphorylation in two different cell lines, thereby providing confirmation of ligand activity.
  • receptor-Fc fusion proteins comprising TGF ⁇ superfamily receptors were screened for binding to human or murine GDF15 that was generated and purified as described in Example 1. These fusion proteins incorporated an IgG1 Fc domain and were either purchased from R&D Systems or generated in-house.
  • TGF ⁇ receptor type II TGF ⁇ receptor type II
  • activin receptor type HA activin receptor type IIB
  • BMP receptor type II BMP receptor type II
  • MIS receptor type II TGF ⁇ receptor type II
  • hGDF15 bound to captured hT ⁇ RII-Fc at 37° C. with an equilibrium dissociation constant (K D ) of 9.56 nM.
  • None of the seven type I receptors (ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7) displayed detectable binding to GDF15 (mGDF15 at 20 nM or 200 nM).
  • Human T ⁇ RII occurs naturally in at least two isoforms—A (long) and B (short)—generated by alternative splicing in the extracellular domain (ECD) ( FIGS. 5 , 6 ).
  • the hT ⁇ RII-hG1Fc fusion protein (R&D Systems) used for the screening described above incorporates the wild-type T ⁇ RII short isoform.
  • the affinity of mGDF15 binding to a fusion protein incorporating the wild-type T ⁇ RII long isoform was found by surface plasmon resonance to be very similar to that of the fusion protein incorporating the T ⁇ RII short isoform (K D s at 37° C.
  • Applicants Having observed general equivalence of these short and long isoforms with regard to GDF15 binding, Applicants then generated a receptor-Fc fusion protein consisting of the wild-type ECD of hT ⁇ RII short (SEQ ID NO: 7) fused at its C-terminus with a human IgG2 Fc domain via a minimal linker.
  • amino acid position numbering with regard to variants based on the T ⁇ RII short and long isoforms refers to the corresponding position in the native precursors, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • T ⁇ RII could be used as an inhibitor of GDF15.
  • the fusion protein hT ⁇ RII short (23-159)-hG2Fc was tested in A549 cells transfected with a reporter gene containing a CAGA-12 promoter construct and was found to inhibit hGDF15-induced gene activation in such cells with an IC 50 of 0.15-0.5 nM. Potent inhibition of GDF15 signaling by the hT ⁇ RII short ECD provides additional evidence that T ⁇ RII is the high-affinity receptor for GDF15.
  • GDF15 exhibited no detectable binding to ALK5 under cell-free conditions
  • suppression of endogenous ALK5 mRNA by siRNA methodology markedly reduced mGDF15-mediated signaling in A549 cells (a human pulmonary epithelial cell line) compared to control treatment.
  • suppression of other type I receptors ALK2, ALK3, ALK4, and ALK7 by siRNA methodology failed to alter GDF15-mediated signaling in A549 cells.
  • ALK5 TGF ⁇ receptor type I
  • T ⁇ RII also binds with high affinity to TGF ⁇ 1 and TGF ⁇ 3, native T ⁇ RII-Fc fusion protein affects signaling of these ligands as well as GDF15. While in some therapeutic settings this broader spectrum of ligand binding may be advantageous, in other settings a more selective molecule may be superior. Therefore, Applicants sought polypeptides with enhanced or reduced selectivity for GDF15 by generating fusion proteins comprising variants of human T ⁇ RII ECD.
  • the wild-type hT ⁇ RII short (23-159) sequence shown below served as the basis for five receptor ECD variants listed below (SEQ ID NO: 8-12).
  • a wild type hT ⁇ RII short (23-159) was fused to an Fc portion of IgG2 to generate a novel, base Fc fusion construct. See SEQ ID Nos. 50, 51 and 52, below.
  • Applicants also envision five corresponding variants (SEQ ID NO: 14-17) based on the wild-type hT ⁇ RII long (23-184) sequence shown below (SEQ ID NO: 13), in which the 25 amino-acid insertion is underlined. Note that splicing results in a conservative amino acid substitution (Val ⁇ Ile) at the flanking position C-terminal to the insertion. Sequence relationships among several hT ⁇ RII short variants and their hT ⁇ RII long counterparts are indicated in FIG. 7 .
  • Additional T ⁇ RII ECD variants include:
  • hT ⁇ RII-hFc fusion proteins were generated in which five hT ⁇ RII short variants described above were each fused at their C-terminus (via a minimal linker) to a human IgG2 Fc domain, which has the following amino acid sequence (SEQ ID NO: 19):
  • hT ⁇ RII-hFc fusion proteins comprising alternative Fc domains, including full-length human IgG1 Fc (hG1Fc) (SEQ ID NO: 20, below) and N-terminally truncated human IgG1 Fc (hG1Fc short ) (SEQ ID NO: 21, below).
  • hG1Fc full-length human IgG1 Fc
  • hG1Fc short N-terminally truncated human IgG1 Fc
  • a polypeptide unrelated to an Fc domain could be attached in place of the Fc domain.
  • the selected hT ⁇ RII-hFc protein variants incorporate the TPA leader and have the unprocessed amino-acid sequences shown in SEQ ID NOs: 25, 29, 33, 37, and 41 (see Example 5). Corresponding nucleotide sequences for these variants are SEQ ID NOs: 26, 30, 34, 38, and 42.
  • Selected hT ⁇ RII-hFc variants, each with a G2Fc domain (SEQ ID NO: 19) were expressed in HEK-293 cells and purified from conditioned media by filtration and protein A chromatography. Purity of samples for reporter gene assays was evaluated by SDS-PAGE and Western blot analysis.
  • This protein was expressed from a construct including a TPA leader sequence, as shown below (SEQ ID NO:52). Dotted underline denotes leader, and solid underline denotes linker.
  • SEQ ID NO: 52 1 51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101 TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151 FSEEYNTSNP D TGGG VECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV 201 TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV 251 HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT 301 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK 351 LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK
  • the nucleic acid sequence encoding SEQ ID NO:52 is shown below:
  • a reporter gene assay in A549 cells was used to determine the ability of hT ⁇ RII-hFc variants to inhibit activity of GDF15, TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3.
  • This assay is based on a human lung carcinoma cell line transfected with a pGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17: 3091-3100) as well as a Renilla reporter plasmid (pRLCMV) to control for transfection efficiency.
  • the CAGA motif is present in the promoters of TGF ⁇ -responsive genes (for example, PAI-1), so this vector is of general use for factors signaling through SMAD2 and SMAD3.
  • A549 cells (ATCC®: CCL-185TM) were distributed in 48-well plates at 6.5 ⁇ 10 4 cells per well.
  • EMEM Eagle's minimum essential medium
  • BSA medium was removed, and cells were incubated overnight at 37° C. with a mixture of ligands and inhibitors prepared as described below.
  • test articles Serial dilutions of test articles were made in a 48-well plate in a 200 ⁇ l volume of assay buffer (EMEM+0.1% BSA). An equal volume of assay buffer containing the test ligand was added to obtain a final ligand concentration equal to the EC50 determined previously.
  • Human GDF15 and murine GDF15 were generated in-house (see above), while human TGF ⁇ 1, human TGF ⁇ 2, and human TGF ⁇ 3 were obtained from PeproTech. Test solutions were incubated at 37° C. for 30 minutes, then 250 ⁇ l of the mixture was added to all wells. Each concentration of test article was determined in duplicate.
  • This assay was used to screen receptor fusion protein variants for potential inhibitory effects on cell signaling by T ⁇ RII ligands. Consistent with previous reports concerning wild-type T ⁇ RII short -Fc and T ⁇ RII long -Fc (del Re et al., J Biol Chem 279:22765, 2004), none of the variants tested were able to inhibit TGF ⁇ 2, even at high concentrations. However, hT ⁇ RII-hFc variants unexpectedly showed differential inhibition of cellular signaling mediated by GDF15, TGF ⁇ 1, and TGF ⁇ 3.
  • the T ⁇ RII short (23-159/D110K)-G2Fc variant Compared with wild-type T ⁇ RII short (23-159)-G2Fc, the T ⁇ RII short (23-159/D110K)-G2Fc variant exhibited potent inhibition of GDF15 but loss of inhibition of TGF ⁇ 1 and greatly reduced inhibition ( ⁇ 50 fold) of TGF ⁇ 3 (see table below).
  • Position 110 is located in the “hook” region of T ⁇ RII (Radaev et al., J Biol Chem 285:14806, 2010) but has not been suggested to confer selectivity among the recognized T ⁇ RII ligands TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3.
  • this variant displays a profile of differential ligand inhibition in which GDF15 is inhibited most potently, TGF ⁇ 1 least potently, and TGF ⁇ 3 to an intermediate degree.
  • IC 50 mGDF15 hTGF ⁇ 1 hTGF ⁇ 3 (35 (640 (270 Construct ng/ml) pg/ml) pg/ml) Full-length T ⁇ RII short (23-159)- ⁇ 0.12 1.73 0.14 wild-type ECD G2Fc Full-length T ⁇ RII short (23-159/ ⁇ 0.7 ND ⁇ 6.9 ECD with D110K)-G2Fc (>73.6) D110K substitution ND, not determined
  • T ⁇ RII short (29-159)-G2Fc and T ⁇ RII short (35-159)-G2Fc displayed a greatly diminished ability to inhibit TGF ⁇ 3 but an undiminished (N′ ⁇ 6) or only slightly diminished (N′ ⁇ 12) ability to inhibit GDF15 compared to T ⁇ RII short (23-159)-G2Fc (wild-type). Effects of N-terminal truncation on inhibition of TGF ⁇ 1 compared to wild-type were intermediate in magnitude. Thus, these two variants exhibit a profile of differential ligand inhibition in which GDF15 is inhibited most potently, TGF ⁇ 3 least potently, and TGF ⁇ 1 to an intermediate degree.
  • IC 50 hGDF15 hTGF ⁇ 1 hTGF ⁇ 3 (70 or 112 (640 (270 Construct ng/ml) pg/ml) pg/ml) Full-length T ⁇ RII short (23-159)- 0.14-0.53 0.52 0.37 wild-type ECD G2Fc N′ ⁇ 6 ECD T ⁇ RII short (29-159)- 0.40 2.05 ND G2Fc (>7.5) N′ ⁇ 12 ECD T ⁇ RII short (35-159)- 0.92 2.51 ND G2Fc (>7.5) ND, not determined
  • T ⁇ RII short (23-153/N70D)-G2Fc displayed greatly diminished ability to inhibit TGF ⁇ 1 and virtually undiminished ability to inhibit TGF ⁇ 3 compared to T ⁇ RII short (23-153)-G2Fc.
  • Effects of N70D substitution on inhibition of GDF15 compared to both T ⁇ RII short (23-153)-G2Fc and wild-type were intermediate in magnitude.
  • the C-terminally truncated variant with N70D substitution exhibits a profile of differential ligand inhibition in which TGF ⁇ 3 is inhibited most potently, TGF ⁇ 1 least potently, and GDF15 to an intermediate degree.
  • IC 50 hGDF15 hTGF ⁇ 1 hTGF ⁇ 3 (70 (640 (270 Construct ng/ml) pg/ml) pg/ml) Full-length T ⁇ RII short (23-159)- 0.14 ND ND wild-type ECD G2Fc C′ ⁇ 6 ECD T ⁇ RII short (23-153)- 0.18 2.62 0.14 G2Fc C′ ⁇ 6 ECD T ⁇ RII short (23-153/ 2.42 17.7 0.28 with N70D N70D)-G2Fc substitution
  • T ⁇ RII long ECD counterparts of these T ⁇ RII short ECD variants will exhibit similar ligand selectivity.
  • a C′ ⁇ 6 truncated ECD (such as SEQ ID NOs: 11 and 16 for the T ⁇ RII short and T ⁇ RII long isoforms, respectively) can be used as a base sequence for T ⁇ RII short or T ⁇ RII long in which to introduce mutations and N-terminal truncations.
  • Item 1 shows the amino acid sequence of hT ⁇ RII short (23-159/D110K)-hG2Fc (SEQ ID NO: 25). Double underline indicates D110K substitution. Dotted underline denotes leader, and solid underline denotes linker.

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